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Session III – Homologous Replacement/Gene Targeting

Optimising gene repair strategies in cell culture

Gene Therapy volume 9, pages 700–702 (2002)Cite this article

Abstract

Gene repair, the precise modification of the genome, offers a number of advantages over replacement gene therapy. In practice, gene targeting strategies are limited by the inefficiency of homologous recombination in mammalian cells. A number of strategies, including RNA-DNA oligonucleotides (RDOs) and short DNA fragments (SDFs), show promise in improving the efficiency of gene correction. We are using GFP as a reporter for gene repair in living cells. A single base substitution was introduced into GFP to create a nonsense mutation (STOP codon, W399X). RDOs and SDFs are used to repair this mutation episomally in transient transfections and restore green fluorescence. The correction efficiency is determined by FACS analysis. SDFs appear to correct GFP W399X in a number of different cell lines (COS7, A549, HT1080, HuH-7), although all at a similar low frequency (∼0.6% of transfected cells). RDOs correct only one of our cell lines significantly (HT1080-RAD51), these cells overexpress the human RAD51 gene; the bacterial RecA homologue. The GFP W399X reporter is a fusion gene with hygromycin (at the 5′ end), this has allowed us to make stable cell lines (A549, HT1080) to study genomic correction. Initial studies using our correction molecules show only low efficiencies of genomic repair (∽10−4). Polyethylenimine (PEI) is used to deliver RDOs and SDFs into mammalian cells in culture for our study. We have used fluorescently labelled RDOs and SDFs to study the effectiveness of this process. FACS analysis of transfected nuclei implied efficient delivery (>90%) both with SDFs and RDOs. However, confocal fluorescence microscopy suggests that a large proportion of the complexed RDO/SDF appears to remain outside the nucleus (or attached to the nuclear membrane). On the basis of these data we are assessing new delivery methods and factors that may alter recombination status to optimise gene repair.

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References

  1. Bigger B, Coutelle C . Perspectives on gene therapy for cystic fibrosis airway disease BioDrugs 2001 15: 615–634

    Article  CAS  Google Scholar 

  2. Davis JC, Geddes DM, Alton EW . Gene therapy for cystic fibrosis J Gene Med 2001 3: 409–417

    Article  Google Scholar 

  3. Flotte TR, Laube BL . Gene therapy in cystic fibrosis Chest 2001 120: 1245–1315

    Article  Google Scholar 

  4. Griesenbach U, Alton EWFW . Recent progress in gene therapy for cystic fibrosis Curr Opin Mol Ther 2001 3: 385–389

    CAS  PubMed  Google Scholar 

  5. Yáñez RJ, Porter ACG . Therapeutic gene targeting Gene Therapy 1998 5: 149–159

    Article  Google Scholar 

  6. Richardson PD, Kren BT, Steer CJ . Targeted gene correction strategies Curr Opin Mol Ther 2001 3: 327–337

    CAS  PubMed  Google Scholar 

  7. Kunzelmann K et al. Gene targeting of CFTR DNA in CF epithelial cells Gene Therapy 1996 3: 859–867

    CAS  PubMed  Google Scholar 

  8. Goncz KK, Kunzelmann K, Xu Z, Gruenert DC . Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments Hum Mol Genet 1998 7: 1913–1919

    Article  CAS  Google Scholar 

  9. Kapsa R et al. In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement Hum Gene Ther 2001 12: 629–642

    Article  CAS  Google Scholar 

  10. Goncz KK et al. Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR Gene Therapy 2001 8: 961–965

    Article  CAS  Google Scholar 

  11. Gamper HB et al. The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts Nucleic Acids Res 2000 28: 4332–4339

    Article  CAS  Google Scholar 

  12. Gamper HB et al. A plausible mechanism for gene correction by chimeric oligonucleotides Biochem 2000 39: 5808–5816

    Article  CAS  Google Scholar 

  13. Igoucheva O, Alexeev V, Yoon K . Targeted gene correction by small single-stranded oligonucleotides in mammalian cells Gene Therapy 2001 8: 391–399

    Article  CAS  Google Scholar 

  14. Liu L, Rice MC, Kmiec EB . In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides Nucleic Acids Res 2001 29: 4238–4250

    Article  CAS  Google Scholar 

  15. Yoon K, Cole-Strauss A, Kmiec EB . Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA/DNA oligonucleotide Proc Natl Acad Sci USA 1996 93: 2071–2076

    Article  CAS  Google Scholar 

  16. Kren BT, Cole-Strauss A, Kmiec EB, Steer CJ . Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide Hepatology 1997 25: 1462–1468

    Article  CAS  Google Scholar 

  17. Colosimo A et al. Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments Mol Ther 2001 3: 178–185

    Article  CAS  Google Scholar 

  18. Inoue N et al. Introduction of single base substitutions at homologous chromosomal sequences by adeno-associated virus vectors Mol Ther 2001 3: 526–530

    Article  CAS  Google Scholar 

  19. Cole-Stauss A et al. Targeted gene repair directed by chimeric RNA-DNA oligonucleotides in a mammalian cell-free extract Nucleic Acids Res 1999 27: 1323–3330

    Article  Google Scholar 

  20. Igoucheva O, Peritz AE, Levy D, Yoon K . A sequence-specific correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using lacZ shuttle system Gene Therapy 1999 6: 1960–1971

    Article  CAS  Google Scholar 

  21. Rice MC, Bruner M, Czymmek K, Kmiec EB . In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae Mol Microbiol 2001 40: 857–868

    Article  CAS  Google Scholar 

  22. Yáñez RJ, Porter ACG . Gene targeting is enhanced in human cells overexpressing hRAD51 Gene Therapy 1999 6: 1282–1290

    Article  Google Scholar 

  23. Thorpe PH, Stevenson BJ, Porteous DJ . Functional correction of episomal mutations with short DNA fragments and RNA-DNA oligonucleotides J Gene Med 2002 4: 195–204

    Article  CAS  Google Scholar 

  24. Lukacsovich T, Waldman BC, Waldman AS . Efficient recruitment of transfected DNA to a homologous chromosomal target in mammalian cells Biochim Biophys Acta 2001 1521: 89–96

    Article  CAS  Google Scholar 

  25. Cavazzana-Calvo M et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease Science 2000 288: 669–672

    Article  CAS  Google Scholar 

  26. Krause DS et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell Cell 2001 105: 369–377

    Article  CAS  Google Scholar 

  27. Borthwick DW et al. Evidence for stem-cell niches in the tracheal epithelium Am J Respir Cell Mol Biol 2001 24: 662–670

    Article  CAS  Google Scholar 

Download references

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  1. Medical Genetics Section, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh, UK

    P Thorpe, B J Stevenson & D J Porteous

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  1. P Thorpe
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  2. B J Stevenson
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  3. D J Porteous
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Correspondence to P Thorpe.

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Thorpe, P., Stevenson, B. & Porteous, D. Optimising gene repair strategies in cell culture. Gene Ther 9, 700–702 (2002). https://doi.org/10.1038/sj.gt.3301750

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